Abstract
BackgroundClostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.ResultsThe C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.ConclusionMultiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of C. botulinum neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.
Highlights
Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases
A comprehensive search was conducted for potential distant homologues of CNTs, starting with a genomic analysis of C. botulinum strain A, followed by a more general search involving additional clostridial and eukaryotic species
Multiple independent links to collagenase-related sequences were identified, including the detected similarities involving the upstream flagellin gene (CBO0798) in the BoNT/A neurotoxin gene cluster, distant BLAST hits to collagenase-related domains, and detected structural similarities to the collagen NC4 domain
Summary
Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Clostridial neurotoxins (CNTs) are the most poisonous biological toxins known and molecular agents of botulism and tetanus neuroparalytic diseases [1]. Due to their extreme toxicity and potential threat as bioterrorism agents, they are listed as Category A agents by the Centers for Disease Control and Prevention along with other deadly agents such as anthrax. As demonstrated by the scattered phyletic distribution of neurotoxin-producing clostridia [10] and the patterns of sequence similarity between different neurotoxin gene clusters [11], CNT genes appear to have undergone significant lateral transfer between different species of Clostridium. The occurrence of lateral transfer is supported by the discovery of plasmid-encoded neurotoxin genes in numerous C. botulinum strains [12], as well as the existence of putative insertion sequences flanking the neurotoxin gene cluster [13]
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